Staphylococcus Biofilm Cultivation and Detachment Protocol

Standardized methods are essential for reliable research outcomes. Here you can find a cultivation and detachment protocol for staphylococcal biofilms. This protocol focusses on streamlined high-throughput sample preparation using UIP400MTP multi-well plate sonicator for efficient, high-throughput biofilm detachment in 96-well plates. The protocol also contains key steps for biofilm cultivation, washing, and visualization, with a focus on minimizing variability and ensuring reproducibility.

Staphylococcus Biofilm and Antibiotic Research

Staphylococcus biofilms play a critical role in persistent infections due to their resistance to antibiotics and immune responses. Biofilm formation provides a protective environment for bacteria, making infections difficult to treat. Research into biofilms often focuses on understanding their formation, behavior, and susceptibility to antimicrobials, with an emphasis on high-throughput methods to streamline experimental workflows.
The UIP400MTP multi-well plate sonicator offers a significant advantage in biofilm research by enabling the rapid and efficient detachment of biofilms from 96-well plates. This device provides uniform ultrasonic energy to all wells, ensuring consistent results while minimizing variability.

Protocol for Staphylococcus Biofilm Cultivation and Detachment

Below, we guide you with a step-by-step instruction through the process of cultivating and detaching a staphylococcus biofilm. As exemplary analytical step, we show you how to quantify spectrophotometrically the cultivated biomass by crystal violet staining.

Cultivation of Staphylococcus Biofilm

Materials Required:

• Sterile flat-bottomed 96-well polystyrene tissue culture-treated microtiter plates with lids
• Tryptic Soy Broth (TSB) with 0.25% glucose
• Biological safety cabinet

Steps:

1. Prepare a sterile working environment in a biological safety cabinet to minimize contamination.
2. Add TSB containing 0.25% glucose to the microtiter plate wells. TSB without glucose does not generally support biofilm formation and should only be used as a control if required.
3. Inoculate the wells with bacterial strains prepared as described below:
4. Prepare bacterial suspensions, ensuring no preexisting cell clusters by homogenizing the suspensions using sonication or by breaking up clusters with a 23-gauge needle and brief vortexing.
5. Seal the plate with its lid and incubate under conditions optimal for biofilm formation (e.g., 37°C for 24 hours).
6. Perform the experiment in triplicate for each bacterial strain (three wells per strain) to ensure reliability.
7. Allocate six wells per plate for negative controls. Up to 30 strains can be tested per 96-well plate.

Biofilm Visualization and Washing

1. Following incubation, discard the medium carefully to avoid disturbing the biofilm.
2. Wash each well four times with physiological saline to remove planktonic bacteria.
3. Inspect the bottom of the wells for white patches indicative of biofilm presence.

Biofilm Detachment Using the Multi-Well Plate Sonicator UIP400MTP

Device Setup and Parameters:

• UIP400MTP multi-well plate sonicator
• Operating settings: 60% amplitude, cycle mode with 60 seconds ON / 30 seconds OFF

Steps:

1. Place the washed microtiter plate onto the UIP400MTP platform.
2. Sonicate the samples at recommended settings (60% amplitude, 60 seconds ON, 30 seconds OFF). Adjust the settings to bacteria strain.
3. Initiate the sonication process to detach the biofilm. The ultrasonic waves disrupt the biofilm matrix, releasing the adherent bacteria.
4. The UIP400MTP ensures uniform exposure across all wells for consistent detachment results.

Analytical Step: Quantification of Detached Staphylococcus Biofilm Biomass Using Crystal Violet (CV)

Materials Required:

• 0.1% crystal violet (CV) solution
• 95% ethanol or 30% acetic acid (for solubilization)
• Microplate reader capable of reading at 570 nm
• Sterile microtiter plates for staining

Steps:

1. Preparation of Staining Plate: Transfer 100 µL of the detached biofilm suspension from each well of the sonicated plate into corresponding wells of a clean, sterile 96-well microtiter plate. This ensures a clear and uniform environment for staining.
2. Staining the Detached Biofilm: Add 150 µL of 0.1% crystal violet solution to each well containing the detached biofilm suspension. Gently pipette to ensure even mixing of the biofilm suspension and crystal violet.
3. Incubation: Allow the plate to incubate at room temperature for 15 minutes to enable the crystal violet to stain the biomass effectively.
4. Washing: After incubation, carefully discard the crystal violet solution from the wells without disturbing the biomass. Wash each well three times with sterile physiological saline to remove unbound stain.
5. Drying: Let the plate air dry at room temperature or under a sterile airflow hood. Avoid heating, as this may alter results.
6. Solubilization: Add 200 µL of 95% ethanol (or 30% acetic acid, depending on standard lab practices) to each well to solubilize the bound crystal violet. Mix gently by pipetting or shaking the plate for 10 minutes at room temperature.
7. Measurement: Measure the optical density (OD) of the solubilized crystal violet solution at 570 nm using a microplate reader.
8. Data Analysis: Subtract the mean OD570 value of negative controls (wells with TSB but no bacterial inoculum) from experimental wells to account for background staining. Record and analyze the data.

Note: Perform the experiment in triplicate for each condition to ensure reproducibility. Ensure proper handling of crystal violet and ethanol, adhering to safety and disposal protocols.
 

The key benefits of the UIP400MTP at a glance:

• High-Throughput Processing: Designed specifically for multi-well plates, allowing you to process multiple samples simultaneously.
• Uniform Ultrasonic Distribution: Ensures equal ultrasonic intensity across wells, providing consistent results across all samples.
• Use Any Standard Plate: The UIP400MTP can handle any standard multi-well plates, Petri dishes and tube racks. No expensive proprietary plates required!
• User-Friendly Interface: Easy to set up and control, making it an excellent tool for increasing lab productivity. Programmable settings and automation facilitate process standardization!